Multiplex Single-Nucleotide Microbial Genome Editing Achieved by CRISPR-Cas9 Using 5′-End-Truncated sgRNAs

Multiplex genome editing with CRISPR-Cas9 offers a cost-effective solution for time and labor savings. However, achieving high accuracy remains a challenge. In an Escherichia coli model system, we achieved highly efficient single-nucleotide level simultaneous editing of the galK and xylB genes using the 5′-end-truncated single-molecular guide RNA (sgRNA) method. Furthermore, we successfully demonstrated the simultaneous editing of three genes (galK, xylB, and srlD) at single-nucleotide resolution. To showcase practical application, we targeted the cI857 and ilvG genes in the genome of E. coli. While untruncated sgRNAs failed to produce any edited cells, the use of truncated sgRNAs allowed us to achieve simultaneous and accurate editing of these two genes with an efficiency of 30%. This enabled the edited cells to retain their lysogenic state at 42 °C and effectively alleviated l-valine toxicity. These results suggest that our truncated sgRNA method holds significant potential for widespread and practical use in synthetic biology.


MATERIALS AND METHODS
Strains and Culture Conditions.

Plasmid Construction.
The plasmids used in experiments related to multiplex genome editing are listed in Table S2.Primers used for plasmid construction are listed in Table S3.Single sgRNA plasmid was constructed in the same way as described in our previous study. 3pHK470 was created by inserting the sgRNA cassette downstream of the spectinomycin resistance gene of pTargetF (provided generously by Sheng Yang; Addgene plasmid #62226).pSR020 was constructed by replacing the spectinomycin resistance gene of pSR001 with the chloramphenicol resistance gene amplified from pACYC184. 4Triple sgRNA plasmid was generated using pHK470 and pSR020 as templates and primers with target recognition sequence overhang.For effective ligation of the three fragments, the incubation time was extended to 8 hours while recovering after heat shock transformation.All plasmids were constructed with Gibson Assembly Master Mix (NEB, Cat.No. E2611, MA, USA), and the sequence of the sgRNA cassette was confirmed through Sanger sequencing.

Construction of Target-Knockout Strains.
To construct a Cas9-harboring strain in which a single target gene (galK or xylB) was knocked out, the P1vir phage (provided generously by Sankar Adhya at the NIH) was used to introduce mutations to HL026 via standard P1 transduction.The P1 lysates of JW0740 and JW3536 from the Keio collection 2 were used.To knock out both galK and xylB, the kanamycin resistance gene was removed from SR005 using pCP20, 5 and the P1 lysate of JW0740 was used to transduce SR008 as a recipient strain.After P1 transduction, colony PCR was performed to verify whether each target gene was substituted with the kanamycin resistance (Km R ) cassette.The identified colony was purified three times on LB agar plates containing kanamycin and used in the experiment.Primers used for colony PCR are listed in Table S3.

Oligonucleotide-Directed Multiplex Genome Editing Procedure.
As in a previous study, pHK463 expressing λ Beta protein was used to increase the single-strand oligonucleotidemediated recombineering efficiency and cas9-integrated E. coli MG1655 was used for genome editing. 6For genome editing, E. coli MG1655 was made into a competent cell after overexpression of λ Beta protein of pHK463 and Cas9 in the chromosome.At 30 °C, E. coli was cultured until OD600nm = 0.4.Then, L-arabinose was added at a final concentration of 1 mM and incubated for 3 hours.The cultured cells were harvested, washed twice with 10% glycerol, resuspended, and stored at −80 °C.Mutagenic oligonucleotides (100 or 500 pmol each) and sgRNA plasmids (200 ng) were electroporated into the cells under conditions of 25 μF, 200 Ω, and 1.8 kV using a 0.1 cm electroporation cuvette (Bio-Rad).Thereafter, 950 mL of SOC medium was immediately added and recovered at 30 or 37 °C and 180 rpm.After 1 hour of recovery, cells were spread on LB or MacConkey agar (BD Difco, Cat.No. 281810, USA) plate containing chloramphenicol (12.5 μg/mL) or spectinomycin (50 or 75 μg/mL) and incubated at 30 or 37°C for 16 hours.MacConkey agar was used to check the fermentation type by adding D-galactose (0.5%, CAS No. 59-23-4), D-xylose (0.5%, CAS No. 58-86-6), or Dsorbitol (0.5%, CAS No. 50-70-4).Mutagenic oligonucleotides used for multiplex genome editing are listed in Table S4.

Calculation of Negative Selection and Editing Efficiency.
Surviving colonies (white + red colonies) were counted to determine whether target cleavage by the CRISPR-Cas9 system was successful.Editing efficiencies were calculated by counting the number of red and white colonies in MacConkey agar ([white colonies] / [white + red colonies]).After three repetitions of the experiment, two to four white colonies were randomly selected and confirmed by Sanger sequencing for each gene (galK, xylB, or srlD).For cI and ilvG editing, editing efficiency was calculated as the ratio of multiplex-edited colonies for both genes among 10 randomly selected colonies.Edited colonies were confirmed by Sanger sequencing of each two genes.Primers used for PCR amplification and Sanger sequencing are listed in Table S3.genes using 5′-end-truncated sgRNAs.After multiplex editing by transforming dual 5′-end-truncated sgRNA plasmid (pSR054) and mutagenic oligonucleotides, 10 colonies were randomly selected from LB agar.(A) MG1655 (λ -), HL012 (λ cI 857 ), and 10 randomly selected colonies were grown on LB agar.λ cI 857 cells undergo lysis at 42 ℃.*Edited λ cI WT cells can grow at 42 ℃.(B) BL21(DE3) (ilvG WT ), MG1655 (ilvG -), and the same 10 randomly selected colonies were grown on M9 D-glucose (0.4%) medium.L-valine (0.1 mM) was added if needed.# Unedited ilvG -cells cannot grow on M9 agar containing D-glucose and L-valine due to L-valine toxicity.

S16
Supplementary Table S1.Strains used in this study.

S19
Supplementary Table S3.Primers used in this study (continued).

Table S3 .
Primers used in this study.

Table S4 .
Mutagenic oligonucleotides used in this study. S21Supplementary

Table S4 .
Mutagenic oligonucleotides used in this study (continued).
-Deleted nucleotides are indicated in parenthesis.